Phenotyping floral traits and essential oil profiling revealed considerable variations in clonal selections of damask rose (Rosa damascena Mill.)

Damask rose (Rosa damascena Mill.) is a high-value aromatic plant species belonging to the family Rosaceae. It is being cultivated throughout the world for rose essential oil production. Besides its higher demand in the aromatic and cosmetic industry, the essential oil obtained has many pharmacological and cytotoxic activities. The primary concern of growers with the available varieties of damask rose is short flowering duration, low essential oil content and unstable yield. Thus, there is a requirement for developing new stable varieties with higher flower yield and essential oil content. The present study evaluated the variations in the flower yield parameters, essential oil content, and essential oil compounds in different clonal selections of damask rose. These clonal selections have been developed through a half-sib progeny approach from commercially available varieties 'Jwala' and 'Himroz.' The fresh flower yield varied from 629.57 to 965.7 g per plant, while the essential oil content ranged from 0.030–0.045% among the clonal selections. The essential oil profiling via gas chromatography–mass spectrometry revealed significant variations in the essential oil compounds. Acyclic monoterpene alcohols citronellol (20.35–44.75%) and geraniol (15.63–27.76%) were highest, followed by long-chain hydrocarbons, i.e., nonadecane (13.02–28.78%). The clonal selection CSIR-IHBT-RD-04 was unique in terms of the highest citronellol content (44.75%) and citronellol/geraniol (C/G) ratio of 1.93%. This selection has the potential use as a parental line in future genetic improvement programs of damask rose to achieve higher yield and better quality of rose essential oil.


Material and methods
Experimental material and location. The present investigations were conducted on four newly developed damask rose selections made in half-sib progeny lines. The lines were derived from commercial varieties 'Jwala' and 'Himroz. ' The lines are being maintained clonally in the rose germplasm repository along with check varieties (Jwala and Himroz) at CSIR-Institute of Himalayan Bioresource Technology, Palampur (1320 m above mean sea level, 32°68'N, 76°38'E). The rose germplasm repository at CSIR-IHBT maintains different cultivated and wild Rosa species from India and worldover, which were introduced through the Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICAR-NBPGR), Regional station at Phagli, Shimla (Himachal Pradesh), India. The location is the mid-hills zone (Zone-II) of Himachal Pradesh (India), which represents sub temperate and humid climate with a mean annual rainfall of ~ 2500 mm, mainly during the monsoon season (July-September). The rose germplasm repository at CSIR-IHBT maintains different cultivated and wild Rosa species from India and the world. The study was carried out on five-year-old plants of each clonal line over two consecutive years (2021 and 2022). A basal fertilizer dose calculated as 120 kg nitrogen (N), 60 kg phosphorus (P), and 40 kg potassium (K) per ha was applied during both years. All the agronomic practices were followed as per the recommendations. The experiment was set up in Randomized Complete Block Design (RCBD), having 1.5 m of plant spacing between rows and 0.75 m within rows. The number of replications for each clonal line is four. It is also to confirm that all methods were performed in accordance with the relevant guidelines and regulations.
Observation for yield-related floral traits. Data was recorded on four random competitive plants per clonal line in each replication. These plants were tagged, and observations were repeated during the second year. The morphological parameters recorded in both years were the number of flower-bearing shoots/plant, flower weight (g), flower diameter (cm), petal number, petal length (cm), petal width (cm), petal thickness (mm), petal weight/flower (g), flower frequency/plant/day, flower number/plant, flower yield/plant, number of flowering days. Data were recorded daily during the flowering period (April third week to May last week). The weather data during the flowering period of the damask rose for both the experimental years (2021 and 2022) is given in Fig. 1.
Essential oil extraction. The flowers were picked manually during morning hours (6:00-9:00 am) to avoid loss of aroma compounds for both years. Fresh flowers (1 kg) of each clonal line were harvested, and essential oil extraction was done by hydro-distillation for four hours (in triplicate), using Clevenger-type apparatus of five liters distillation system. The ratio of flower to water used for extraction was 1:2 (w/v). The essential oil obtained from each sample was measured, and oil content (w/w) was depicted in percentage (%) on a fresh weight basis. The moisture content in essential oil was removed using sodium sulfate (anhydrous). The essential oil is collected in a glass vial and kept in a refrigerator (4-6 °C) till further chemical characterization. After that, gas chroma- www.nature.com/scientificreports/ tography-flame ionization detector (GC-FID) and GC-mass spectrometry (GC-MS) analysis were used for the chemical characterization of essential oil compounds present in rose oil.
GC-MS/GC analysis of essential oil. GC-MS characterization of damask rose essential oil was performed using a Shimadzu GC-MS QP2010 gas chromatograph attached to a flame ionization detector (FID). The essential oil was analyzed over SH-RX-5Si/MS capillary column, Shimadzu Asia Pacific, USA (30 m × 0.25 mm × 0.25 μm film thickness) attached to the gas chromatograph. The GC-MS analysis was carried out with the same conditions reported earlier [22][23][24] . The retention indices (RI) for all the chemical compounds were calculated using homologous series of n-alkanes C9-C24 (SUPELCO, Sigma-Aldrich). The retention indices were calculated for every GC-MS spectra peak to identify the compounds. The calculated retention indexes were compared with Adams tabulated indexes 25 stored in the NIST-mass spectral database 26 . After identifying the essential oil compounds, the next step was the quantification performed through GC analysis. The GC analysis was carried out using Shimadzu GC 2010 gas chromatograph attached to a flame ionization detector (FID). The analysis was performed on the same capillary column described above. The instrument was operated with the same conditions reported earlier [22][23][24] . Finally, individual compounds were quantified using the peak area percentage of the chromatogram. Also, the mass spectral fragment patterns of the chemical compounds were compared with those reported in the literature.

Statistical analysis.
The phenotypic data of floral traits and damask rose clonal lines yield were recorded for two consecutive years. The analysis of variance (ANOVA) was performed to test the performance of clonal lines during both years. The Variations among clonal lines were determined using the F-test (comparing genotypes' mean with check varieties). Data for the morphological traits were analyzed using multivariate clustering following Euclidean similarity co-efficient with Past 1.40 software 27 . The Eigenvalues of characters loading were calculated to find out the effect of characters on the clustering. Principal component analysis was done to identify key characters which differentiate the clonal lines into distinct groups. The correlation studies were executed to explore the relationship between floral traits using the Pearson correlation matrix. The correlation coefficient (r) for different essential oil compounds was calculated using OP STAT 28 , and the matrix was prepared using Past 1.40 software.

Results and discussion
Variations in the yield-related floral traits. Significant variations were observed among the damask rose clonal lines for floral traits studied during both years. Based on the F-value significant differences among lines were obtained for flower-bearing shoots, flower frequency/plant/day, flower number per plant and flower yield per plant ( Table 1). The number of flowers per plant is the most important component determining flower  (Table 2). Based on PCA analysis using pooled data from two years, the flower-bearing shoots were the key principal component (PC1), explaining 96.833% of the variance. In contrast, flower weight was the 2nd principal component (PC2), explaining 2.974% of variance that influenced the differentiation and clustering of clonal lines, whereas all other floral traits had low eigenvalue loadings. However, using variance-covariance matrix scatter plot of principal components, plants of clones CSIR-IHBT-RD-01, CSIR-IHBT-RD-03, CSIR-IHBT-RD-04 and Jwala grouped independently, while those of Himroz and CSIR-IHBT-RD-02 grouped in the same cluster (Fig. 2).
The maximum flower yield was obtained in clonal selection CSIR-IHBT-RD-04 (944.07 g/plant in the first year and 931.05 g/plant in the second year), followed by CSIR-IHBT-RD-01 (880.08 g/plant in 2021 and 881.19 g/   (Table 1).
Overall, based on the mean value of both studied years, CSIR-IHBT-RD-04 (937.56 g/plant) was superior, followed by CSIR-IHBT-RD-01 (880.63 g/plant) and check variety Jwala (752.93 g/plant). Check variety Himroz (663.33 g/plant) was inferior to CSIR-IHBT-RD-03 (819.19 g/plant) but performed better than CSIR-IHBT-RD-02 (640.32 g/plant). Based on mean flower frequency per plant daily, peaks were observed almost weekly for the clonal lines in both years. The mean flower frequency/plant/day was the least at the initiation and fag end of the flowering season. It reached a maximum after about 20 days of flower initiation during both years (maximum flower frequency/plant/day) based on the pooled average of clonal lines as 17.95 in 2021 and 18.42 in 2022 (Fig. 3).
In the case of CSIR-IHBT-RD-04, there was consistently higher flower frequency/plant/day from 22-32 days of the flowering period compared to other clonal lines, which differentiated the line based on its phenology. An earlier report based on principle component analysis suggested that parents with a higher fresh weight of Flower, number of petals per flower and bud width can be used for hybridization during the genetic improvement program of damask rose 29 . Correlation and regression analysis. Correlation studies were performed based on the two-year pooled data of the floral traits to identify significant variation. Based on the correlation matrix (Table 3)   www.nature.com/scientificreports/ A high correlation among traits indicates a strong association among the traits, whereby one trait influences the expression of the other. Accordingly, a regression equation was established between independent variables, i.e., flower frequency/plant/day, flower-bearing shoots and flower number/plant) and dependent variable, i.e., flower yield per plant, to establish the association based on a second-degree polynomial relationship. Figure 4A, B and C displayed a comparatively low statistical correlation between these independent variables to the flower yield with a coefficient of determination (R 2 ) ranging from 0.59 to 0.60. The inter-relationships among flower frequency/plant/day, flower-bearing shoots and flower number/plant were also tested using regression analysis.
The flower frequency/plant/day and flower-bearing shoots (R 2 = 0.88, Fig. 4D) showed better relationships among each other. Flower number/plant showed a comparatively higher association with Flower bearing shoots (R 2 = 0.91, Fig. 4E). In comparison, the flower number/plant showed a stronger second-degree polynomial relationship (y = 37.512x + 2.2006, R 2 = 0.97, Fig. 4F) with flower frequency/plant/day. Flower yield in damask rose is an economically crucial trait after essential oil quality and accurate floral phenotyping is critical to identify potential selections and maximize production.
Essential oil yield (w/w %). The essential oil yield of the four clonal selections (CSIR-IHBT-RD-01 to CSIR-IHBT-RD-04) and two check varieties (Himroz and Jwala) maintained at CSIR-IHBT Palampur are depicted in Fig. 5. The essential oil content varies from 0.030 to 0.045% of the fresh flower weight in kilogram during both the experimental years (2021 and 2022). The important physico-chemical properties of the damask rose essential oil is depicted in Fig. 6. Based on the comparison of four selections in terms of rose oil yield, the clonal selection CSIR-IHBT-RD-04 showed a higher percentage of essential oil (0.040% in 2021 and 0.042% in www.nature.com/scientificreports/ 2022) compared to others clonal selections. However, a t-test using standard deviation shows significant variations in essential oil yield (0.45%) in the check variety Himroz for both years. Usually, the yield of essential oil in damask rose from the western Himalayas is reported to be 0.017 to 0.051 5 . However, through appropriate agronomical interventions, the essential oil content may reach a high of 0.056% under the acidic conditions of the western Himalayas 33 . In a recent study from Iran, the essential oil content was reported to be 0.03-0.04% 1 . The genetic architecture of the plant species might be another reason for the variation in essential oil content during the present study. Clonal selection has the advantage of maintaining the homogenous grade of essential oil   www.nature.com/scientificreports/ for industrial use 24 . Accordingly, evaluating clonal lines is necessary for selecting superior clones with a higher essential oil yield for a specific region.

GC-MS based essential oil profiling of clonal lines and check varieties indicates chemotypic distinctions.
A comparative study of essential oil composition was carried out by gas chromatography-mass spectrophotometry (GC-MS) to understand the chemotypic distinctions with respect to essential oil composition in four clonal lines (CSIR-IHBT-RD-01 to CSIR-IHBT-RD-04), and two check varieties (Himroz and Jwala) of damask rose. Hydro distillation of fresh flowers led to obtaining uncolored to yellowish essential oil. Overall, twenty-six compounds were identified in the essential oil via GC-MS analysis, which accounted for 97.04 to 99.48% of the total essential oil profile. The essential oil components were grouped into oxygenated monoterpenes (36.62 to 70.05%), oxygenated sesquiterpenes (2.80 to 6.57%), sesquiterpene hydrocarbon (2.63 to 6.40%) and aliphatic hydrocarbons (19.94 to 55.68%). The retention time and indices of all the essential compounds are summarized in Table 4. The analysis of composition data suggested that the oxygenated monoterpenes and aliphatic hydrocarbon were the major fractions in the essential oil. The representative GC-MS chromatogram of the major compound of damask rose essential oil is given in Fig. 7. Diverse chemotypic distinctions were obtained in the essential oil composition. Eighteen compounds were present in all the clonal lines and check varieties studied (Table 5). Significant variations were observed based on a t-test using standard deviation for essential oil components among the clonal lines and check varieties for both years. Based on the mean value of the component, the highest content of cis-rose oxide (1.67%) and trans-rose oxide (1.26%) were observed for check variety Himroz during 2021 that was statistically at par with the clonal selection CSIR-IHBT-RD-04. However, during 2022 significant variations were observed for check varieties Himroz (1.16 and 1.43%) and Jwala (1.14 and 1.34%) for cis-rose and trans-rose oxides, respectively.

CSIR-IHBT-RD-01 CSIR-IHBT-RD-02 CSIR-IHBT-RD-03 CSIR-IHBT-RD-04 Himroz
The variations in essential oil composition observed in the present study are possibly due to the genotypic response of different selections to changing weather conditions during the flowering period for both years. The meteorological conditions, such as the maximum temperature, minimum temperature, and relative humidity at the evening, were comparatively higher during 2022. In contrast, total rainfall, relative humidity at the morning and sunshine hours were comparatively higher during 2021. The clonal selection CSIR-IHBT-RD-04 exhibited significantly higher citronellol, eugenol and pentadecane content, including citronellol/geraniol ratio (C/G ratio) 2022 compared to 2021. Compared to other lines, a positive response of CSIR-IHBT-RD-04 for citronellol content in essential oil was obtained at a relatively high-temperature regime (upto 30 °C in 2022, compared to 26.50 °C in 2021) and dry climate (68.35 mm rainfall and 48.15% relative humidity in 2022, compared to 108 mm rainfall and 62.0% relative humidity in 2021) during the flowering period. A similar type of difference in essential oil compounds has earlier been observed, confirming the influence of ecological and environmental conditions 35 , genetic factors 36 and post-harvesting on the biosynthesis of secondary metabolites.
Our results for major essential oil compounds of damask rose align with the previous reports where the acyclic monoterpene alcohols (citronellol and geraniol) and long-chain hydrocarbons (n-nonadecane and heneicosane) were the major components 1,37 . The acyclic monoterpene alcohol, i.e., citronellol, is responsible for the rose-like aroma of the essential oil 1,38 . A higher amount of citronellol in the essential oil indicates higher quality 1 . Earlier studies reported the highest amount of 42% citronellol in the essential oil of damask rose from the western Himalayan conditions 39 . The most important/sensitive indicator of damask rose oil odor quality is the citronellol/geraniol ratio (C/G ratio) between 1.25 and 1.30 1,38 . In our present study, the C/G ratio for essential oil samples varies from 1.18 to 1.93%. The clonal selection CSIR-IHBT-RD-04 was superior in flower yield and flower frequency/plant/day compared to other clonal lines. The essential oil content was higher in CSIR-IHBT-RD-04 during both years compared to other clonal lines except for check variety Himroz. Based on the GC-MS profiling of the essential oil, CSIR-IHBT-RD-04 captures unique chemotypic diversity in terms of the highest citronellol content (37.20% in 2021 and 44.75% in 2022). The C/G ratio was also significantly higher in CSIR-IHBT-RD-04 during 2022. The clonal line CSIR-IHBT-RD-04 has also been registered with the Indian Council of Agricultural Research-Plant Germplasm Registration Committee, New Delhi, under accession number IC0635435, INGR20105 as new germplasm based on its peculiar characteristics.

Conclusion
The present study investigated the variations for floral traits, the essential oil profile of the four clonal lines, and two check varieties of damask rose. The study was undertaken to identify superior clonal selection for high yield and quality oil composition. The selection CSIR-IHBT-RD-04 was superior in flower yield and had higher flower frequency/plant/day than other clonal lines. The essential oil content was also higher in CSIR-IHBT-RD-04 when compared with other clonal lines except for check variety Himroz. Based on GC-MS profiling of essential oil, CSIR-IHBT-RD-04 displays unique chemotypic diversity in terms of the highest citronellol content and citronellol/geraniol (C/G) ratio, which is the chief indicator of high quality. Clonal selection CSIR-IHBT-RD-04 may be used as a parental line in the hybridization program for genetic improvement of damask rose.